The present invention relates generally to an enzymatic process for the determination of free fatty acids, and more particularly to a process for the estimation of free fatty acids with the use of acyl coenzyme A (hereinafter referred to as acyl-CoA) synthetase and acyl-CoA oxidase, in which a sequence of the reactions involved are accelerated by the addition of myokinase, thereby to permit rapid and precise determination of free fatty acids.
In general, method for the determination of free fatty acids has depended upon chemical processes including, organic solvent extraction, which are troublesome to handle. Accordingly, there is a strong demand for a more simplified process.
Recently, however, an enzymatic method for the determination of free fatty acids in serum with the use of acyl-CoA synthetase has been developed (see Japanese Laid-Open Publication No. 52-17085).
This type of determination is based on a sequence of enzymatic systems wherein the acyl-CoA synthetase is allowed to act on the free fatty acids in the presence of adenosine triphosphate (hereinafter called ATP) to yield adenosine monophosphate (hereinafter termed AMP). Formation of AMP is then followed as the production of pyruvic acid proceeds by the action of myokinase and pyruvate kinase. The amount of pyruvic acid formed is principally in proportion to the guantities of the free fatty acids. As serum usually contains ATP-decomposing enzymes such as ATPases and phosphatases, more or less, the ATP in the measuring system breaks down under the action of these enzymes into adenosine diphosphate (ADP), which is measured as pyruvic acid. Another apparent reaction owing to the endogeneous pyruvic acid takes place in this method. These defects lead to a problem in that the resulting value is unreliable.
In addition to the above-mentioned type of quantification, the assay methods utilizing other enzymatic systems are introduced. In these methods, acyl-CoA formed from free fatty acid by the action of acyl-CoA synthetase is measured as the production of hydrogen peroxide by using an acyl-CoA oxidase. The hydrogen peroxide formed is followed colorimetrically by the action of peroxidase 4-aminoantipyrine and phenol.
The method of such a type has a great advantage that the apparent reaction is not deleteriously influenced, even when ATPases, phosphatases and pyruvic acid are present in the serum. It is found, however, that there is still much left to be desired from a practical viewpoint.
When the acyl-CoA synthetase acts on the free fatty acids in the presence of ATP, the resultant AMP hinders the formation of acyl-CoA. Therefore, the free fatty acids in the serum are not completely converted to acyl-CoA. Even though a given amount of acyl-CoA oxidase is then made to act on the acyl-CoA to yield hydrogen peroxide, the serum free fatty acids are not completely converted to hydrogen peroxide; hence, the obtained measurement of the fatty acids is lower than the true value.
To eliminate this defect due to a low reaction rate, it is required to apply either a higher concentration of the acyl-CoA synthetase or the measuring time must be considerably increased. Such an additional operation spoils the commercial value of the process.
As a result of extensive studies carried out with a view to obviating these defects, the present inventors have found that a sequence of the reactions involved can be promoted by converting AMP formed under the action of acyl-CoA synthetase into ADP by the action of myokinase. By introducing this conception, i.e., removal of AMP from the reaction systems, it is possible to withdraw restrictions placed upon the formation of acyl-CoA by AMP and to force the formation of acyl-CoA to rapid completion.